Controlling Confinement and Topology to Study Collective Cell Behaviors.

Confinement and substrate topology strongly affect the behavior of cell populations and, in particular, their collective migration. In vitro experiments dealing with these aspects require strategies of surface patterning that remain effective over long times (typically several days) and ways to control the surface topology in three dimensions. Here, we describe protocols addressing these two aspects.

Traveling Pulses for a Two-Species Chemotaxis Model.

Mathematical models have been widely used to describe the collective movement of bacteria by chemotaxis. In particular, bacterial concentration waves traveling in a narrow channel have been experimentally observed and can be precisely described thanks to a mathematical model at the macroscopic scale. Such model was derived in [1] using a kinetic model based on an accurate description of the mesoscopic run-and-tumble process.

A multiscale study of bacterial proliferation modes within novel E. coli@Si(HIPE) hybrid macrocellular living foams.

For the first time the study at various length scales of E. coli proliferation modes within Si(HIPE) inorganic macrocellular foams is proposed. Both qualitatively and semi-quantitatively, bacterial proliferation within the foam is not homogeneous and is directly linked to the random distribution of Si(HIPE) macroscopic cells.

Biochemical perturbations of the mitotic spindle in Xenopus extracts using a diffusion-based microfluidic assay.

A microfluidic device is a powerful tool to manipulate in a controlled manner at spatiotemporal scales for biological systems. Here, we describe a simple diffusion-based assay to generate and measure the effect of biochemical perturbations within the cytoplasm of cell-free extracts from Xenopus eggs. Our approach comprises a microliter reservoir and a model cytoplasm that are separated by a synthetic membrane containing sub-micrometric pores through which small molecules and recombinant proteins can diffuse.

Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells.

The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established.

Stimuli-responsive topological change of microstructured surfaces and the resultant variations of wetting properties.

It is now well established that topological microstructures play a key role in the physical properties of surfaces. Stimulus-induced variations of topological microstructure should therefore lead to a change in the physical properties of microstructured responsive surfaces. In this paper, we demonstrate that roughness changes alter the wetting properties of responsive organic surfaces.

Automated velocity mapping of migrating cell populations (AVeMap).

Characterizing the migration of a population of cells remains laborious and somewhat subjective. Advances in genetics and robotics allow researchers to perform many experiments in parallel, but analyzing the large sets of data remains a bottleneck. Here we describe a rapid, fully automated correlation-based method for cell migration analysis, compatible with standard video microscopy.

Modeling E. coli tumbles by rotational diffusion. Implications for chemotaxis.

The bacterium Escherichia coli in suspension in a liquid medium swims by a succession of runs and tumbles, effectively describing a random walk. The tumbles randomize incompletely, i.e.

Directional persistence of chemotactic bacteria in a traveling concentration wave.

Chemotactic bacteria are known to collectively migrate towards sources of attractants. In confined convectionless geometries, concentration "waves" of swimming Escherichia coli can form and propagate through a self-organized process involving hundreds of thousands of these microorganisms. These waves are observed in particular in microcapillaries or microchannels; they result from the interaction between individual chemotactic bacteria and the macroscopic chemical gradients dynamically generated by the migrating population.

Thermal separation: interplay between the Soret effect and entropic force gradient.

Thermophoresis, the Soret effect, depletes a high concentration of a polyethylene glycol polymer solution from the hot region and builds a concentration gradient. In such a solution, solutes of small concentration experience thermophoresis and polyethylene glycol concentration-dependent restoring forces. We report that by using focused laser heating and varying the polyethylene glycol concentration one observes geometrical localizations of solutes like DNA and RNA into patterns such as a ring.

Orientation and polarity in collectively migrating cell structures: statics and dynamics.

Collective cell migration is often characterized by the spontaneous onset of multicellular protrusions (known as fingers) led by a single leader cell. Working with epithelial Madin-Darby canine kidney monolayers we show that cells within the fingers, as compared with the epithelium, are well oriented and polarized along the main finger direction, which suggests that these cells actively migrate. The cell orientation and polarity decrease continuously from the tip toward the epithelium over a penetration distance of typically two finger lengths.

Traction forces exerted by epithelial cell sheets.

Whereas the adhesion and migration of individual cells have been well described in terms of physical forces, the mechanics of multicellular assemblies is still poorly understood. Here, we study the behavior of epithelial cells cultured on microfabricated substrates designed to measure cell-to-substrate interactions. These substrates are covered by a dense array of flexible micropillars whose deflection enables us to measure traction forces.

Effect of surface pattern on the adhesive friction of elastomers.

We present experimental results for the friction of a flat surface against a hexagonally patterned surface, both being made of PolyDiMethylSiloxane. We simultaneously measure forces of range 10 mN and observe the contact under sliding velocities of about 100 μm/s. We observe adhesive friction on three different pattern heights (80, 310, and 2100 nm).

Mathematical description of bacterial traveling pulses.

The Keller-Segel system has been widely proposed as a model for bacterial waves driven by chemotactic processes. Current experiments on Escherichia coli have shown the precise structure of traveling pulses. We present here an alternative mathematical description of traveling pulses at the macroscopic scale.

Velocity fields in a collectively migrating epithelium.

We report quantitative measurements of the velocity field of collectively migrating cells in a motile epithelium. The migration is triggered by presenting free surface to an initially confluent monolayer by using a microstencil technique that does not damage the cells. To avoid the technical difficulties inherent in the tracking of single cells, the field is mapped using the technique of particle image velocimetry.

Strength dependence of cadherin-mediated adhesions.

Traction forces between adhesive cells play an important role in a number of collective cell processes. Intercellular contacts, in particular cadherin-based intercellular junctions, are the major means of transmitting force within tissues. We investigated the effect of cellular tension on the formation of cadherin-cadherin contacts by spreading cells on substrates with tunable stiffness coated with N-cadherin homophilic ligands.

Micron-sized main-chain liquid crystalline elastomer actuators with ultralarge amplitude contractions.

Responsive surfaces composed of cylindrical or square micrometer-sized thermoresponsive pillars made of thiol-ene nematic main-chain liquid crystalline elastomers (LCEs) are produced by replica molding. The individual pillars behave as microactuators, showing ultralarge and reversible contractions of around 300-400% at the nematic to isotropic phase transition. The nematic main-chain LCE microactuators described here present contractions as large as the best macroscopic systems reported in the literature.

E. Coli and oxygen: a motility transition.

The motility of Escherichia coli is correlated with oxygen concentration. We show that oxygen penetrating into an anaerobic sample induces the coexistence of two domains of motile and nonmotile bacteria. This coexistence generates a bacterial accumulation at the border that propagates slowly with a constant velocity.

Collective migration of an epithelial monolayer in response to a model wound.

Using an original microfabrication-based technique, we experimentally study situations in which a virgin surface is presented to a confluent epithelium with no damage made to the cells. Although inspired by wound-healing experiments, the situation is markedly different from classical scratch wounding because it focuses on the influence of the free surface and uncouples it from the other possible contributions such as cell damage and/or permeabilization. Dealing with Madin-Darby canine kidney cells on various surfaces, we found that a sudden release of the available surface is sufficient to trigger collective motility.

Forced detachment of immersed elastic rubber beads.

We investigate the strength of adhesion and the dynamics of detachment of elastic beads (Young's modulus E approximately 1 MPa) adhering to a horizontal solid surface in a viscous liquid. The beads are initially compressed on the surface. Their unbinding is imposed by fast vertical stretching (above a certain threshold value).

Efficiency of a self-aminoacylating ribozyme: effect of the length and base-composition of its 3' extension.

Variants of a previously described small self-aminoacylating ribozyme are tested in order to uncover the potentialities of a 3' extension responsible for the esterification. The base-composition and the length of this specific part of the ribozyme are investigated. Very short extensions can still reach the active site, reflecting the small persistence length of RNA.

Adhesion enhancement through micropatterning at polydimethylsiloxane-acrylic adhesive interfaces.

Adhesion at polydimethylsiloxane (PDMS)-acrylic adhesive interfaces is shown to be enhanced through micropatterning of the PDMS substrate. By varying the geometry of the patterns (groves and hexagonal arrays of pillars of micrometer sizes, obtained through soft lithography techniques) and comparing rigid and deformable substrates, the respective roles of the geometry and the size and flexibility of the pattern features on the level of adhesion have been analyzed. For cylindrical pillars, two regimes are clearly identified: for a relatively low aspect ratio (h/r < 3, with h and r, respectively, the height and the radius of the pillars), soft patterned substrates are more efficient than rigid ones at increasing adhesion, pointing out the role of the elastic energy associated with the deformation of the pattern that is lost when the adhesive detaches from the substrate.